Highly infectious diseases originating from bacterial (e.g., anthrax) or viral (e.g. Ebola) pathogens with high mortality rates pose high risks, includin epidemic outbreaks and hostile acts using these pathogens as biological weapons. The ability to detect and respond rapidly at the point of care is essential for dealing with these threats. Despite increased research activity, there is currently no established point-of-care system for these pathogens due to the shortcomings of the current detection approaches, including lack of speed (culture), lack of accuracy (antigen tests), and lack of simplicity (polymerase chain reaction (PCR), in large part due to the need to amplify the genetic target material). Our long-term goal is to develop point-of-care biomedical devices using optofluidics - the combination of integrated optics and microfluidics on a single chip-scale system. The objective of this application is to demonstrate and validate a Hybrid, Integrated Molecular Analysis System (HIMAS) that is suitable for differential point-of-care diagnosis of category A pathogens. Our central hypothesis is that this can be accomplished by combining two powerful microfluidic and optical technologies that are optimized for sample processing and amplification-free detection in separate chip layers. During the initial R21 phase, our objectives will be accomplished by the following specific aims: (1) Introduce a new spectral target multiplexing approach using interferometric waveguide structures; (2) Introduce a new hybrid optofluidic system composed of a glass microfluidic layer and a silicon optical layer; (3) Validate the platform for differential diagnostics of hemorrhagic fever viruses using clinical samples. In a subsequent R33 phase, we will build on these innovations by developing a portable prototype system that can rapidly distinguish between 14 weaponizable hemorrhagic fever viruses without the need for target amplification, starting from a whole blood patient sample. The innovative contributions of the proposed approach are: (i) interferometric excitation using multi-mode interferometer (MMI) waveguides for spectral, spatial, and combinatorial target multiplexing; (ii) introduction of a new planar optofluidic system with layers optimized individually for sample processing and amplification-free nucleic acid analysis. The proposed work is significant because it overcomes the critical barriers to developing a point-of-care system for PCR-free, differential diagnostics o biodefense pathogens and other viral and bacterial threats to human health.